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. 2021 Jun 7;148(11):dev198044. doi: 10.1242/dev.198044

Fig. 1.

Fig. 1.

‘Ventral targeting’ events during regeneration of PLM axon correlate with functional restoration. (A) The experimental set-up for the axotomy of PLM neuron [labeled with Pmec-7::GFP (muIs32) and Pmec-4::mCherry::RAB-3 (tbIs227)] at the L4 stage, and follow-up steps to correlate functional recovery with regrowth patterns. The PTRI was measured at 3 h post-axotomy to measure the functional drop after injury. Similarly, the PTRI was measured at 24 h or 48 h post-axotomy to evaluate the extent of functional recovery, following which the axon regrowth pattern in the same animal was imaged. The confocal images of two major regrowth patterns are shown. The red arrow indicates the site of injury. The worm illustration describes how PLM neuron is connected to the AVA and PVC interneurons. UNC-9::GFP [shrEx435] was used to label the electrical synapse. ‘Cm’ represents the co-injection GFP-reporter in coelomocyte. (B) The PTRI values corresponding to the ‘fusion’ and ‘non-fusion’ events at 24 h and 48 h post-axotomy at L4 stage. N (independent replicates)=4-12. (C) The depth-coded pseudocolored z-stacked confocal images and illustrations of various regrowth patterns. The arrowheads indicate the regrowth towards the ventral direction and red arrows indicate the injury site. (D) The quantification of the RI expressed as PTRI at 24 h post-axotomy/PTRI at 3 h post-axotomy for different regrowth trajectories. The dashed green line denotes an RI value of 1. N=6-8. (E,F) Confocal images of PLM expressing Pmec-7::GFP::RAB-3 (jsIs821) and Pmec-4::mScarlet (shrEx209). (E) The large puncta of GFP::RAB-3 can be observed at the tip of the ventrally targeted axon along the VNC. (F) The enrichment of GFP::RAB-3 (arrowheads) at the end of the synaptic branch also referred to as developmental synapse (DS). Insets show an enlarged view of the region inside the yellow dashed box. (G) The ratio of GFP::RAB-3 intensity to the intensity of mScarlet from the rectangular ROIs shown in E and F. N=3-5. Red arrows indicate the site of injury. The yellow and white arrowheads represent the GFP::RAB-3 enrichment along the VNC in the case of the ventrally targeted axon and the developmental synapse, respectively. Data are mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (ANOVA with Tukey's multiple comparisons test).